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Propofol anesthesia is reduced in phospholipase C-related inactive protein

type-1 knockout mice

Yoshikazu Nikaido, Tomonori Furukawa, Shuji Shimoyama, Junko Yamada, Keisuke

Migita, Kohei Koga, Tetsuya Kushikata, Kazuyoshi Hirota, Takashi Kanematsu,

Masato Hirata, Shinya Ueno

Affiliations

Hirosaki University Graduate School of Medicine, Hirosaki, Japan (Y.N.); Department

of Neurophysiology (Y.N., T.F., K.K., S.U.), and Anesthesiology (Y.N., T. Kushikata,

K.H.), Hirosaki University Graduate School of Medicine, Hirosaki, Japan; Research

Center for Child Mental Development, Hirosaki University Graduate School of

Medicine, Hirosaki, Japan (S.S., S.U.); Department of Biomedical Sciences, Division

of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences,

Hirosaki, Japan (J.Y.); Department of Drug Informatics, Faculty of Pharmaceutical

Sciences, Fukuoka University, Fukuoka, Japan (K.M.); Department of Cellular and

Molecular Pharmacology, Division of Basic Life Sciences, Institute of Biomedical and

Health Sciences, Hiroshima University, Hiroshima, Japan (T. Kanematsu); Laboratory

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of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University,

Fukuoka, Japan (M.H.); Fukuoka Dental College, Fukuoka, Japan (M.H.).

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Running title: PRIP-1 is involved in propofol-induced anesthesia

Corresponding author

Shinya Ueno, MD, PhD

Department of Neurophysiology, Hirosaki University Graduate School of Medicine, 5

Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

Tel. and Fax: +81-172-39-5138

E-mail: shinyau@hirosaki-u.ac.jp

Number of text pages: 38

Number of tables: 2

Number of figures: 4

Number of references: 47

Number of words in the Abstract: 182

Number of words in the Introduction: 419

Number of words in the Discussion: 1164

Abbreviations

Phospholipase C-related inactive protein type-1 (PRIP-1); loss of righting reflex

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(LORR): loss of tail-pinch withdrawal response (LTWR); okadaic acid (OA); protein

phosphatase (PP); total anesthesia score (TAS).

Section: Behavioral Pharmacology

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Abstract

The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics.

Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R

phosphorylation and membrane trafficking. In this study, we investigated the role of

PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate,

and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO)

mice by measuring the latency and duration of loss of righting reflex (LORR) and loss

of tail-pinch withdrawal response (LTWR). The effect of okadaic acid (OA), a protein

phosphatase 1/2A inhibitor, pretreatment on propofol- and etomidate-induced LORR

was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR

induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1

could determine the potency of the anesthetic action of propofol. Pretreatment with

OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA

injection enhanced phosphorylation of cortical the GABAA-R β3 subunit in PRIP-1 KO

mice. These results suggest that PRIP-1-mediated GABAA-R β3 subunit

phosphorylation might be involved in the general anesthetic action induced by

propofol but not by etomidate or pentobarbital.

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Introduction

Phospholipase C-related inactive protein type-1 (PRIP-1) is an inositol

1,4,5-trisphosphate binding protein, homologous to phospholipase C-δ1 but

catalytically inactive (Kanematsu et al, 1992; Kanematsu et al, 1996; Matsuda et al,

1998). PRIP-1 has various binding partners including GABA type A receptor

(GABAA-R) β1–3 subunits (Terunuma et al. 2004), GABAA-R-associated protein

(Kanematsu et al, 2002), protein phosphatases 1 (PP1) and 2A (PP2A; Yoshimura et

al, 2001; Kanematsu et al, 2006; Yanagihori et al, 2006), and Akt protein kinase (Fujii

et al, 2010). PRIP-1 acts as a bridge between the γ2 GABAA-R subunit and

GABAA-R-associated protein, and facilitates GABAA-R membrane trafficking

(Kanematsu et al, 2002; Mizokami et al, 2007). PRIP-1 binds to GABAA-R β subunits

as a scaffold protein of PP1/2A, which regulate the phosphorylation level of β

subunits (Terunuma et al, 2004; Kanematsu et al, 2006; Yanagihori et al, 2006;

Kanematsu et al, 2007). PP1/2A dephosphorylate the β subunit and induce receptor

endocytosis mediated by clathrin adaptor protein (AP2) and clathrin

(Comenencia-Ortiz et al, 2014). PRIP-1 inactivates PP1 activity and reduces PP2A

activity (Terunuma et al, 2004; Kittler et al, 2005; Kanematsu et al, 2007; Sugiyama et

al, 2012). Phosphorylation of PRIP-1 itself and lack of PRIP-1 liberates the active

form of PP1 and further activates PP2A (Terunuma et al, 2004; Yanagihori et al,

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2006; Sugiyama et al, 2012). Our previous studies revealed that PRIP-1 knockout

(PRIP-1 KO) mice had reduced extrasynaptic GABAergic transmission (tonic

inhibition) in the hippocampus, cerebral cortex, and spinal cord (Migita et al, 2011;

Zhu et al, 2012). Furthermore, the effects of diazepam on GABAergic extrasynaptic

transmission were markedly reduced in PRIP-1 KO mice compared with wild-type

(WT) mice. Consequently, the anticonvulsant action of diazepam was suppressed in

PRIP-1 KO mice (Zhu et al, 2012). However, it remains unknown how PRIP-1 affects

anesthetic action through the regulation of GABAergic transmission.

Most intravenous anesthetics enhance the inhibitory action of GABAA-R

(Rudolph and Antkowiak, 2004). Extrasynaptic GABAergic transmission is more likely

to contribute to general anesthesia than synaptic transmission (Semyanov et al,

2004; Bieda et al, 2004; Belelli et al, 2009; Bieda et al, 2009; Kubo et al, 2009; Herd

et al, 2014). The phosphorylation state of GABAA-R is thought to determine its

localization and activity in the synaptic or extrasynaptic membrane (Terunuma et al,

2004; Abramian et al, 2010; Comenencia-Ortiz et al, 2014).

Here, we investigated whether PRIP-1 deficiency influences the anesthetic

action of three intravenous anesthetics, propofol, etomidate, and pentobarbital, and

whether PRIP-1-mediated GABAA-R phosphorylation states are involved in the

anesthetic action of these anesthetics.

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Methods

Animals

We used adult male PRIP-1 KO (n = 223) and WT (C57BL/6; n = 184) mice, aged 12–

18 weeks and weighing 25–35 g. Animals were group-housed at 24 ± 2°C under a

12-h light/dark cycle (lights on at 8:00 am) and food and water were available ad

libitum. The experimental procedures used in this study complied with the guidelines

for animal research issued by the Physiological Society of Japan and Hirosaki

University School of Medicine, and all efforts were made to minimize the number of

animals used and their suffering.

Drugs

For behavioral studies, mice received propofol (Maruishi Pharmaceuticals, Osaka,

Japan), etomidate (Tokyo Chemical Industry, Tokyo, Japan), or pentobarbital

(Somunopentyl; Kyoritsu Seiyaku, Tokyo, Japan) intraperitoneally (i.p.) at a volume of

10 µL/g body weight. The vehicle for propofol and etomidate was 20% intralipid fluid

solution (Fresenius Kabi, Tokyo, Japan). Pentobarbital was diluted with 0.9% saline

(Otsuka Pharmaceuticals, Tokyo, Japan). PP1/2A inhibitor okadaic acid (OA) sodium

salt (Wako Pure Chemical Industries, Tokyo, Japan) was dissolved in 0.9% saline

(0.01 mg/mL) and injected (10 µL/g body weight i.p.) 30 min before administration of

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propofol or etomidate. The dose of OA used in this study did not cause cyanosis,

dyspnea, or diarrhea (Tubaro et al, 2003). No hypnotic or analgesic effects were

observed after intralipid or saline injection in either genotype (n = 4 per group; data

not shown). For western blotting, mice were injected (10 µL/g body weight i.p.) with

OA (0.01 mg/mL) or saline. Other animals received OA (1 pg/5 µL) or vehicle (saline,

5 µL) intracerebroventricularly (i.c.v.) under 1–3% isoflurane (Escain; Pfizer, Tokyo,

Japan) for general anesthesia and lidocaine (Xylocaine jelly 2%; AstraZeneca, Osaka,

Japan) for local surface anesthesia, as described previously (Maeda et al, 2005).

Thirty min after i.p. injection, or 25 min after i.c.v. injection, mice were euthanized with

pentobarbital (150 mg/kg i.p.) and brain tissue was collected (n = 3 per group).

Behavioral analysis

Loss of righting reflex (LORR) was used to measure the hypnotic effect of the

anesthetic agents. Immediately after drug injection, each animal was placed in a

chamber (20 × 28 × 15 cm) with an electric heat pad, and the righting reflex was

assessed every 2 min for a maximum of 2 h. Anesthesia was evaluated using the

following scoring system (Irifune et al, 2003): 0, normal righting reflex; 1, righting

within 2 s; 2, righting latency 2–10 s; 3, no righting within 10 s. Anesthetic scores

were determined every 2 min as the median of three trials. Total anesthetic score

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(TAS) was the sum of all anesthetic scores used to evaluate anesthetic efficacy.

Sensitivity to anesthetics was expressed as the percentage of animals to be given a

score of 3 (% LORR). The time from drug administration to a score of 3 was

considered the latency to LORR, and the duration of LORR was measured as the

time between a score of 3 and a subsequent score of 2. When a score of 1 was

followed by two consecutive scores of 0, mice were considered to have recovered

from anesthesia.

Loss of tail-pinch withdrawal response (LTWR) assay was performed to

evaluate immobilization, which is known to be the anesthetic-induced ablation of the

supraspinal nociceptive response to noxious stimuli. A surgical clip (125 g/cm2,

micro-serrefine No 18055-04; Fine Science Tools, Foster City, CA) was placed at the

base of an animal’s tail for 10 s (cutoff time) at 15 min after anesthetic administration.

The nociceptive response was measured by the time (test latency) when the mouse

showed any response to tail-pinch.

Baseline nociceptive response (baseline latency) was measured 30 min before

drug injection. To estimate LTWR, the percentage of maximal possible effect (% MPE)

was evaluated using the formula: % MPE = 100 × (test latency − baseline latency) /

(cutoff time − baseline latency). The magnitude of LTWR was assessed by the area

under the % MPE vs. time curve (AUC) using the trapezoidal method (Maeda et al,

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2005).

Western blotting

Immediately after euthanasia, each mouse was perfused with ice-cold

phosphate-buffered saline (PBS) and the brain removed (Maeda et al, 2005; Zhu et al,

2012). Thick coronal sections of the frontal cortex were made at the level of the

striatum (Paxinos and Franklin, 2001). Total protein extracts and western blotting

were performed as previously described (Ozaki et al, 2012). The crude membrane

fraction was prepared using a previous method with modification (Goebel-Goody et al,

2009; Garcão et al, 2014). Tissue was homogenized in ice-cold homogenization

buffer (pH 6.0) containing the following (in mM): 320 sucrose, 20 Tris-HCl, 0.1 CaCl2,

1 MgCl2, cOmplete™ Protease Inhibitor Cocktail EDTA-free (Roche Diagnostics,

Mannheim, Germany), and PhosSTOP Phosphatase Inhibitor Cocktail (Roche

Diagnostics). The homogenate was centrifuged at 1000 × g for 10 min, and then

supernatant was further centrifuged at 10 000 × g for 20 min to obtain the crude

membrane fraction. The precipitate was dissolved in homogenization buffer

containing 1% SDS and saved as the crude membrane fraction. Solubilized proteins

were isolated from the frontal cortex and equal amounts of protein was separated by

SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride

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membrane (Bio-Rad, Hercules, CA). Membranes were incubated with the following

primary antibodies: anti-GABAA-R β2,3 subunit antibody (Millipore, Temecula, CA);

anti-GABAA-R β3 subunit antibody (Millipore); anti-GABAA-R β3 phospho-Ser408/409

antibody (PhosphoSolutions, Aurora, CO); anti-β-actin antibody (Abcam, Cambridge,

MA); or anti-calnexin antibody (Synaptic Systems, Göttingen, Germany). Membranes

were washed with Tween 20–PBS and incubated with horseradish

peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse IgG (Dako, Cambridge,

UK). Immunoreactive signals were developed with an enhanced chemiluminescence

western blotting detection kit (GE Healthcare, Princeton, NJ) and quantified using a

luminescent image analyzer (LAS-4000; Fujifilm, Tokyo, Japan). Band intensities

were measured using ImageJ software (http://rsbweb.nih.gov/ij/).

Statistics

To estimate the median effective dose (ED50) with 95% confidence, probit analysis

was carried out on the LORR dose–response data for each anesthetic using

generalized linear model function glm with probit link function and dose predict

function dose.p in the R-package MASS (Venables and Ripley, 2002; R Core Team,

2016). TAS expressed as the median (range) was analyzed using the Mann–Whitney

U-test. Data for the latency and duration of LORR and % MPE of LTWR are

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expressed as mean ± SEM. To compare the effects of anesthetics on LORR and

LTWR between genotypes, unpaired t-tests were performed. One-way ANOVA with a

post-hoc Tukey’s test was used to determine the effects of OA on LORR induced by

propofol or etomidate. Unpaired t-tests were used to assess the effects of OA on

membrane expression and phosphorylation levels of the GABAA-R β3 subunit. All

statistical analyses were performed with R version 3.3.2 (R Core Team, 2016). A

value of P < 0.05 was considered significant. To estimate effect size as a benchmark

for assessing the magnitude of differences between WT and PRIP-1 KO mice,

Cohen’s d value for unpaired t-tests and Mann–Whitney U-tests, and Cohen’s f value

for one-way ANOVA were computed using the test function in the R-package

compute.es (Del Re, 2013). Cohen defined a small effect as d = 0.20 or f = 0.10,

medium effect as d = 0.50 or f = 0.25, and a large effect as d = 0.80 or f = 0.40 (Cohen,

1992).

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Results

Effect of PRIP-1 deficiency on sensitivity to propofol, etomidate, and

pentobarbital

First, we determined the ED50 for the number of mice showing LORR (% LORR) with

each anesthetic in WT and PRIP-1 KO mice. Table 1 summarizes LORR and TAS

values at each dose for propofol, etomidate, and pentobarbital in PRIP-1 KO and WT

mice. The dose–response curve of % LORR for each anesthetic is shown in Figure 1.

The ED50 of propofol in PRIP-1 KO mice was greater than that in WT mice (Figure 1A,

left panel). Propofol at the maximum dose of 140 mg/kg could not evoke LORR in all

PRIP-1 KO mice tested. TAS for propofol in PRIP-1 KO mice was lower than that in

WT mice (Table 1). There were no differences between genotypes for etomidate or

pentobarbital ED50 values (Figure 1B and C, left panels).

Effect of PRIP-1 deficiency on induction and maintenance of hypnosis induced

by propofol, etomidate, and pentobarbital

Because the highest dose of each anesthetic in this study was most effective at

inducing a stable LORR occurrence and/or increased anesthetic scores for PRIP-1

KO mice (Table 1; temporal anesthetic score changes after each anesthetic treatment

are shown in Supplemental Figures S1–S3), we estimated the hypnotic potency of

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these anesthetics at the highest doses by measuring the latency and duration of

LORR. Latency to LORR in PRIP-1 KO mice after injection of propofol (140 mg/kg

i.p.) was significantly longer than that in WT mice (Figure 1A, right panel). The

duration of propofol-induced LORR in PRIP-1 KO mice was significantly shorter than

that in WT mice. There were no significant differences in latency or duration of LORR

between genotypes after etomidate (30 mg/kg i.p.; Figure 1B, right panel) or

pentobarbital (40 mg/kg i.p.; Figure 1C, right panel) administration.

Effect of PRIP-1 deficiency on immobilization produced by propofol, etomidate,

and pentobarbital

LTWR was examined to evaluate the immobilizing potency of each anesthetic in WT

and PRIP-1 KO mice. PRIP-1 KO mice showed attenuation of propofol (140 mg/kg

i.p.)-evoked LTWR (% MPE) in comparison to WT mice (Figure 2A). The magnitude

of propofol-induced LTWR (AUC) in PRIP-1 KO mice was significantly smaller than

that in WT mice. There were no significant differences between genotypes in % MPE

and AUC of LTWR produced by etomidate (30 mg/kg i.p.; Figure 2B) or pentobarbital

(40 mg/kg i.p.; Figure 2C).

Hypnotic effect of propofol in PRIP-1 KO mice was rescued by OA pretreatment

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PRIP-1 possesses a binding site for GABAA-R subunits and PP1/2A, and functionally

regulates not only trafficking but also phosphorylation of GABAA-R. We have

electrophysiologically demonstrated dysfunction of the extrasynaptic GABAA-R

response in PRIP-1 KO mice (Migita et al, 2011; Zhu et al, 2012). Therefore, we

examined whether PP1/2A inhibitor rescued the perturbed anesthetic action of

propofol. Pretreatment with PP1/2A inhibitor OA before propofol administration

reduced LORR latency and prolonged LORR duration in PRIP-1 KO mice (Figure 3A),

but did not do the same with etomidate administration (Figure 3B). TAS of propofol

(140 mg/kg i.p.), but not etomidate (30 mg/kg i.p.), increased after OA pretreatment

(0.1 mg/kg i.p.) in PRIP-1 KO mice (Table 2). OA pretreatment did not alter these

values in WT mice. These results indicated that recovery of the hypnotic effect of

propofol in PRIP-1 KO mice was provided by phosphorylation of GABAA-R.

Effect of OA on protein expression of GABAA-R β2,3 subunits and the

phosphorylated β3 subunit in PRIP-1 KO mice

The GABAA-R β3 subunit is thought to be a common target of propofol and etomidate.

Therefore, we performed western blotting to evaluate the effect of OA pretreatment

on phosphorylation states for the cortical GABAA-R β3 subunit. There was less

phosphorylation of the GABAA-R β3 subunit in the frontal cortex of PRIP-1 KO mice

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than in WT mice (Figure 4A). Both i.p. and i.c.v. administration of OA enhanced

GABAA-R β3 subunit phosphorylation without affecting overall levels of the β2,3

subunit in PRIP-1 KO mice. In addition, these OA treatments induced enhancement

of β3 subunit membrane expression and phosphorylation levels (Figure 4B). β3

subunit membrane expression levels in PRIP-1 KO mice were lower than those in WT

mice after saline treatment (Figure 4C). Following i.p. or i.c.v. OA treatments, β3

subunit membrane expression increased in both genotypes. Phosphorylation levels of

the membrane GABAA-R β3 subunit in i.p. and i.c.v. saline-administered PRIP-1 KO

mice tended to be lower than in WT mice (Figure 4D). After i.p. or i.c.v. OA injections,

phosphorylated β3 levels in PRIP-1 KO mice were higher (~2-fold) than those in

saline-injected PRIP-1 KO mice.

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Discussion

In this study, we found that a lack of PRIP-1 attenuated the hypnotic and immobilizing

effects of propofol but not etomidate or pentobarbital. Intraperitoneal pretreatment

with OA rescued hypnotic effect of propofol in PRIP-1 KO mice while OA did not alter

the effect of propofol in WT mice. Furthermore, we confirmed that the

dephosphorylated state of the GABAA-R β3 subunit in PRIP-1 KO mice was

recovered by i.p. OA administration, similar to i.c.v. administration.

Propofol mainly exerts its anesthetic action via the GABAA-R β3 subunit (Jurd

et al, 2003; Belelli et al, 2005; Feng and Macdonald, 2004; Drexler et al, 2009).

Etomidate potentiates GABAA-R containing β3 or β2 subunits (Reynolds et al, 2003;

Belelli et al, 2005; Drexler et al, 2009). Our current and previous studies have shown

that PRIP-1 deficiency reduces β3 subunit membrane expression and induces its

dephosphorylation (Figure 4; Zhu et al, 2012). This is because a lack of PRIP-1

induces PP1/2A activation and AP2-clathrin-mediated internalization (Terunuma et al.

2004; Kittler et al, 2005; Kanematsu et al, 2007). However, in this study, the total

expression level of β2/3 subunits in PRIP-1 KO mice did not seem to differ from that

of WT mice, suggesting that the reduction in β3 subunit membrane expression might

be compensated by the β2 subunit. Propofol affinity for the open state of α1β2γ2L

GABAA-R is approximately 3–7-fold weaker than etomidate (Rüsch et al, 2004; Ruesh

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et al, 2013). In addition to the β3 subunit, α1/4 and β2 GABAA-R subunits may be

responsible for pentobarbital anesthesia (Zeller et al, 2007; Mercado and Czajkowski,

2008). This evidence suggests that non-significant but moderate effective changes of

etomidate- and pentobarbital-anesthesia in PRIP-1 KO mice would be because of

membrane expression of β2-containing GABAA-R. There is a possibility that

downregulation of β3-containing GABAA-R in cell surface attenuate propofol

anesthesia.

Tonic inhibition mediated by extrasynaptic GABAA-R activity is more likely to

be involved in anesthetic action than phasic inhibition mediated by synaptic activity

(Akk et al, 2004; Bieda et al, 2004; Belelli et al, 2009; Bieda et al, 2009; Kubo et al,

2009; Herd et al, 2014). Extrasynaptic GABAA-R activity partly depends on receptor

phosphorylation by various kinases such as protein kinase A and protein kinase C

(Comenencia-Ortiz et al, 2014). For example, protein kinase C-induced

phosphorylation of α4β3 subunits, which mediate tonic inhibition, stabilizes GABAA-R

in the cell surface. Consequently, phosphorylated GABAA-R causes enhancement of

GABA-mediated currents (Abramian et al, 2010). In contrast, dephosphorylation of

the β3 subunit by PP1/2A triggers AP2-clathrin-dependent internalization (Terunuma

et al. 2004; Kittler et al, 2005; Kanematsu et al, 2007), probably resulting in the

reduction of GABA-mediated currents. Our current and previous studies revealed that

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PRIP-1 deficiency reduces expression levels of both total and phosphorylated

GABAA-R β3 subunit in membrane fraction (Figure 4; Terunuma et al, 2004; Zhu et al,

2012). Functionally, PRIP-1 deficiency reduces basal tonic currents, but not phasic

currents, in hippocampal and cortical neurons (Zhu et al, 2012). Therefore, PRIP-1

could modulate extrasynaptic GABAA-R activity by preserving the β3 subunit

phosphorylation state and/or by regulating PP1/2A phosphatase activity. PRIP-1

might be involved in the induction and maintenance of propofol-induced anesthesia

through the trafficking of GABAA-R and phosphorylation state of GABAA-R β3 subunit.

Contrary to our finding that PRIP-1 deficiency induced dephosphorylation of

the GABAA-R β3 subunit in the frontal cortex, another study has reported that

phosphorylation levels of the β3 subunit in the hippocampus in PRIP-1 KO mice are

similar to those of WT mice (Terunuma et al, 2004). This discrepancy may be

because of differences in the brain region of interest or in the experimental methods

used (crude vs. immunoprecipitated protein samples). However, the same study also

reported that, in PRIP-1 KO mice, PP1 rather than PP2A phosphatase activity is

enhanced and PP1/2A inhibitors partially regain phosphorylation and function of the

β3 subunit (Terunuma et al, 2004), which support our i.p. and i.c.v. observations.

Decreased PP1 activity in OA-treated PRIP-1 KO mice is thought to lead to

phosphorylation of the β3 subunit. Inhibition of PP2A function by OA injection may

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also contribute to phosphorylation of the β3 subunit, because OA has a higher affinity

to PP2A than PP1 (Chen et al, 1994). PP2A regulates basal levels of β3 subunit

phosphorylation (Jovanovic et al, 2004). As mentioned above, preservation of β3

subunit phosphorylation increases cell surface expression levels of β3-containing

GABAA-R and GABAergic inhibitory transmissions (Abramian et al, 2010;

Comenencia-Ortiz et al, 2014). OA pretreatment in PRIP-1 KO mice enhanced the

induction and maintenance of hypnosis induced by propofol more effectively than

etomidate, suggesting that enhancement of β3 subunit phosphorylation by blocking

PP1/2A activation increases surface expression of β3-containing GABAA-R in PRIP-1

KO mice. In WT mice, OA pretreatment induced slight, but not significant, increases in

anesthetic duration provided by propofol or etomidate. Inhibition of dephosphorylation

by OA might induce opposite effect of propofol and etomidate in WT mice. Relatively

increased phosphorylation of PRIP-1 by OA may liberate the active form of PP1,

leading to activation of PP2A (Sugiyama et al, 2012). It is possible to inhibit the

hypnotic effect of propofol and etomidate. In contrast, blockade of PP1/2A

phosphatase activity by OA may provide potentiation of propofol and etomidate action

in WT mice. Taken together, in the present study, administration of OA in WT mice

provide slight potentiation of propofol and etomidate action.

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Study limitations

The anesthetics administered i.p. in this study are usually given via intravenous bolus

injection or continuous infusion in humans and large animals, so the current results

from i.p. administration in the mouse should be interpreted with caution. We could not

analyze the effects of PRIP-1 deficiency on membrane expression and

phosphorylation of GABAA-R α1–6, β1/2, γ1–3, and δ subunits. These subunit

complexes play important roles in receptor phosphorylation, phasic/tonic inhibition,

and endocytosis (Belelli et al, 2009; Comenencia-Ortiz et al, 2014). The effects of

phosphorylation on GABAA-R are diverse and appear to be highly dependent on the

subunit composition (Terunuma et al, 2004; Kittler et al, 2005; Yanagihori et al, 2006;

Abramian et al, 2010; Comenencia-Ortiz et al, 2014). Therefore, our current data

would be but a part of the interaction among anesthetics, GABAA-R phosphorylation,

and its regulatory protein PRIP-1. Further studies are required to provide

comprehensive mechanisms behind the effects of PRIP-1 deficiency on anesthetic

action.

Conclusions

We found that the PRIP-1-mediated GABAA-R phosphorylation state is important for

the induction of, and emergence from, propofol-induced anesthesia. Enhancement of

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GABAA-R phosphorylation could potentiate propofol anesthesia. While there might be

various technical difficulties, our data imply the anesthetic and/or therapeutic

possibility of GABAA-R phospho-regulation. The preoperative estimation of the

GABAA-R phosphorylation state and its regulatory protein activity, such as PRIP-1,

PP1/2A, or protein kinase C, in a patient would suggest a better propofol and/or other

anesthetic administration plan to prevent adverse events such as awakening during,

and delayed emergence from, anesthesia. PRIP-1 and other proteins involved in

GABAA-R phosphorylation and membrane trafficking may emerge as novel

candidates for agent-specific regulatory factors in the control of anesthetic action.

Acknowledgement

We are thankful to Noriaki Kawai for technical assistance.

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Authorship contributions

Study design: Yoshikazu, Tetsuya, Kazuyoshi, and Shinya.

Data acquisition: Yoshikazu, Tomonori, and Shuji.

Data analysis: Yoshikazu, Tomonori, Shuji, and Shinya.

Writing of the manuscript: Yoshikazu, Tomonori, Shuji, Junko, Keisuke, Kohei,

Tetsuya, Kazuyoshi, Takashi, Masato, and Shinya.

Approval of final manuscript: Yoshikazu, Tomonori, Shuji, Junko, Keisuke, Kohei,

Tetsuya, Kazuyoshi, Takashi, Masato, and Shinya.

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Footnotes

This work was supported by a Hirosaki University Institutional Research grant; a

Karoji Memorial Fund for Medical Research grant; Japan Society for the Promotion of

Science KAKENHI [Grants 23659735, 24229009, 25670663]; and the Ministry of

Education, Culture, Sports, Science and Technology KAKENHI [Grants 23592242,

24659690].

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Figure legends

Figure 1 Effect of PRIP-1 deficit on hypnosis produced by propofol, etomidate, and

pentobarbital

(A) Dose–response curves for loss of righting reflex (LORR) induced by propofol in

wild-type (WT) and PRIP-1 KO mice. Probit analysis was used to fit curves of ED50

values (solid lines) and 95% confidence intervals (dashed curves). ED50 values were

71.0 mg/kg (95% confidence interval, 69.8–72.2 mg/kg) in WT mice and 115 mg/kg

(114–117 mg/kg) in PRIP-1 KO mice. Significant prolongation of latency to LORR was

observed with administration of propofol (140 mg/kg i.p.) in PRIP-1 KO mice

(unpaired t-test: t15 = 2.95, P < 0.05, Cohen’s d = 1.30). PRIP-1 KO mice emerged

from propofol-induced LORR significantly earlier than WT mice (t15 = 3.16, P < 0.05, d

= 1.80). (B) ED50 values of etomidate were 8.85 mg/kg (8.78–8.91 mg/kg) in WT mice

and 8.85 mg/kg (8.79–8.92 mg/kg) in PRIP-1 KO mice and (C) those of pentobarbital

were 17.5 mg/kg (17.2–17.8 mg/kg) in WT mice and 17.2 mg/kg (16.9–17.5 mg/kg) in

PRIP-1 KO mice. There were no significant differences in response to etomidate (30

mg/kg i.p.) or pentobarbital (40 mg/kg i.p.) between WT and PRIP-1 KO mice

(etomidate: latency, t17 = 1.76, P > 0.05, d = 0.81; duration, t17 = 0.94, P > 0.05, d =

0.43; pentobarbital: latency, t10 = 0.68, P > 0.05, d = 0.36; duration, t10 = 0.41, P >

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0.05, d = 0.22). Group n 5 mice. *P < 0.05 vs. WT mice. Data are mean ± SEM.

Figure 2 Effect of PRIP-1 deficit on immobilization induced by propofol, etomidate,

and pentobarbital

(A) Time course and magnitude of propofol-induced loss of tail-pinch withdrawal

response (LTWR). Significant reduction of percentage of maximal possible effect (%

MPE) and AUC were observed in PRIP-1 KO mice (unpaired t-test: % MPE at 60 min,

t15 = 2.30, P < 0.05, Cohen’s d = 1.12; AUC, t15 = 2.25, P < 0.05, d = 1.10). (B) and

(C) There were no significant differences in LTWR produced by etomidate or

pentobarbital between wild-type (WT) and PRIP-1 KO mice (etomidate: AUC, t15 =

0.82, P > 0.05, d = 0.40; pentobarbital: AUC, t9 = 0.49, P > 0.05, d = 0.27). Group n

6 mice. *P < 0.05 vs. WT mice. Data are mean ± SEM.

Figure 3 Effect of okadaic acid (OA) pretreatment on hypnosis induced by propofol

and etomidate in PRIP-1 KO and wild-type (WT) mice

(A) Latency and duration of propofol-induced loss of righting reflex (LORR).

Pretreatment with OA shortened the latency and extended the duration of LORR in

PRIP-1 KO mice (latency: one-way ANOVA, F3, 26 = 9.13, P < 0.001, Cohen’s f = 1.03;

duration: F3, 26 = 7.14, P < 0.01, f = 0.91). (B) Latency and duration of

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etomidate-induced LORR. There were no significant differences in etomidate-induced

LORR in either genotype after pre-injection with OA (latency: F3, 11 = 2.86, P > 0.05, f

= 0.33; duration: F3, 11 = 0.39, P > 0.05, f = 0.40). Group n 4 mice. Tukey’s post-hoc

comparison: *P < 0.05, Cohen’s d > 1.50, vs. WT mice; †P < 0.05, d > 1.60, vs.

PRIP-1 KO mice pretreated with saline. Data are mean ± SEM.

Figure 4 Western blot analysis of whole cell and membrane fraction of GABAA-R β3

and phosphorylated β3 subunits in i.p. or i.c.v. okadaic acid (OA)-treated wild-type

(WT) and PRIP-1 KO mice

(A) In WT mice, significant differences were not observed in GABAA-R β2,3 and

phosphorylated β3 subunits (P-β3; lanes 1–4). In PRIP-1 KO mice, there were no

changes in total protein levels of GABAA-R β2,3 subunits (lanes 5–8). The amount of

P-β3 increased after i.p. or i.c.v. administration of OA (lane 6, 8) compared with i.p. or

i.c.v. saline (lane 5, 7) administration. β-actin was used as an internal control. (B) In

WT mice, surface β3 and P-β3 expression increased after i.p. or i.c.v. administration

of OA. Surface β3 and P-β3 expression decreased in PRIP-1 KO mice and recovered

by i.p or i.c.v. treatment with OA. Gephyrin was used as an internal control of the

membrane fraction. The band intensity was normalized to Gephyrin. (C) and (D) The

results of western blotting of the membrane fraction are represented in a graph. (C)

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Membrane expression of the β3 subunit in PRIP-1 KO mice significantly decreased

compared with WT mice (i.p. treatment: unpaired t-test, t4 = 4.87, P < 0.05, Cohen’s d

= 3.98; i.c.v. treatment: t4 = 3.81, P < 0.05, d = 3.11). OA treatment induced increases

in surface β3 subunit expression levels in both genotypes (WT: i.p.: t4 = 3.28, P < 0.05,

d = 2.68; i.c.v.: t4 = 1.60, P = 0.18, d = 1.31; PRIP-1 KO: i.p.: t4 = 1.90, P = 0.13, d =

1.55; i.c.v.: t4 = 2.57, P = 0.06, d = 2.10). Slight differences were detected in β3

membrane expression levels between WT and PRIP-1 KO mice administered OA

(i.p.: t4 = 1.82, P = 0.14, d = 1.49; i.c.v.: t4 = 2.78, P < 0.05, d = 2.27). (D) Surface

P-β3 expression in PRIP-1 KO mice tended to be lower than that in WT mice (i.p.: t4 =

0.75, P = 0.49, d = 0.61; i.c.v.: t4 = 6.45, P < 0.05, d = 2.27). OA administration

enhanced membrane P-β3 subunit levels in WT and PRIP-1 KO mice (WT: i.p.: t4 =

1.89, P = 0.13, d = 1.53; i.c.v.: t4 = 0.64, P = 0.56, d = 0.52; PRIP-1 KO: i.p.: t4 = 2.72,

P = 0.05, d = 2.22; i.c.v.: t4 = 2.40, P = 0.07, d = 1.96). Membrane P-β3 subunit levels

were comparable between genotypes treated with OA (i.p.: t4 = 0.93, P = 0.41, d =

0.76; i.c.v.: t4 = 0.25, P = 0.82, d = 0.20). *P < 0.05, Cohen’s d = 2.68, vs. mice treated

with saline; †P < 0.05, d 2.27, vs. WT mice with the same treatments. Data are

mean ± SEM.

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Table 1 Effects of anesthetics on TAS and LORR in WT and PRIP-1 KO mice 1

Anesthetic Dose (mg/kg)

WT PRIP-1 KO

Effect sizec

na TASb na TASb

Propofol 40 0/6 0 (0–3)

0/6 0 (0–21) 0.40

60 3/7 1 (0–25)

0/7 0 (0–3) 1.03

80 4/6 36.5 (0–43)

1/6 16.5 (0–24)* 1.51

100 6/7 52 (0–110)

6/14 7.5 (0–45)* 1.31

120 7/7 97 (14–161)

7/13 21 (6–127)* 0.92

140 7/7 71 (54–149)

10/14 29 (6–79)* 1.87

Etomidate 5 0/6 0 (0–1)

0/6 0 (0–3) 0.07

8 2/10 2.5 (0–81)

2/11 0 (0–41) 0.67

10 7/8 21 (0–78)

8/9 17 (0–86) 0.21

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20 6/6 47 (36–147)

6/6 48 (19–101) 0.05

30 9/9 125 (82–180)

10/10 107.5 (58–180) 0.72

Pentobarbital 10 0/6 0 (0–0)

0/6 3 (0–15) 0.77

15 2/6 11.5 (0–29)

2/5 12 (6–68) 0.64

20 5/7 15 (5–22)

5/7 15 (3–43) 0.17

30 6/6 38.5 (24–46)

9/9 30 (18–43) 0.82

40 6/6 88 (41–136)

6/6 71 (42–164) 0.28

a Denominators represent the number of animals tested in each group. Numerators represent animals that showed LORR. b TAS 2

express the sum of anesthetic scores as median (range). c Effect size (Cohen’s d) for TAS compared with WT mice with the same 3

treatment. *P < 0.05, vs. WT mice with the same treatment (Mann–Whitney U-test). 4

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Table 2 Effects of OA on anesthetic-induced TAS and LORR in WT and PRIP-1 KO mice 5

Anesthetic

Dose

(mg/kg)

OA dose

(mg/kg)

WT PRIP-1 KO

Effect size

na TASb

na TASb

Propofol 140 0 6/6 121 (68–151) 7/8 38.5 (3–99)* 1.59c

140 0.1 6/6 169.5 (106–179) 11/12 148 (30–175)† 0.87c

1.33d

Etomidate 30 0 4/4 112 (110–149) 5/5 110 (89–177) 0.92c

30 0.1 4/4 179 (99–181) 5/5 166 (16–180) 0.70c

0.79d

a Denominators represent the number of animals tested in each group. Numerators represent animals that showed LORR. b TAS 6

express the sum of anesthetic scores as median (range). c Effect size (Cohen’s d) for TAS compared with WT mice with the same 7

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treatment. d Cohen’s d for TAS compared with PRIP-1 KO mice pretreated with OA (0 mg/kg). *P < 0.05, vs. WT mice with the same 8

treatments; †P < 0.05, vs. PRIP-1 KO mice pretreated with OA (0 mg/kg) (Mann–Whitney U-test). 9

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